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. 2013:955:381-99.
doi: 10.1007/978-1-62703-176-9_21.

Tubular crystals and helical arrays: structural determination of HIV-1 capsid assemblies using iterative helical real-space reconstruction

Peijun Zhang  1 , Xin Meng, Gongpu Zhao
Affiliations

Tubular crystals and helical arrays: structural determination of HIV-1 capsid assemblies using iterative helical real-space reconstruction

Peijun Zhang et al. Methods Mol Biol. 2013.

Abstract

Helical structures are important in many different life forms and are well-suited for structural studies by cryo-EM. A unique feature of helical objects is that a single projection image contains all the views needed to perform a three-dimensional (3D) crystallographic reconstruction. Here, we use HIV-1 capsid assemblies to illustrate the detailed approaches to obtain 3D density maps from helical objects. Mature HIV-1 particles contain a conical- or tubular-shaped capsid that encloses the viral RNA genome and performs essential functions in the virus life cycle. The capsid is composed of capsid protein (CA) oligomers which are helically arranged on the surface. The N-terminal domain (NTD) of CA is connected to its C-terminal domain (CTD) through a flexible hinge. Structural analysis of two- and three-dimensional crystals provided molecular models of the capsid protein (CA) and its oligomer forms. We determined the 3D density map of helically assembled HIV-1 CA hexamers at 16 Å resolution using an iterative helical real-space reconstruction method. Docking of atomic models of CA-NTD and CA-CTD dimer into the electron density map indicated that the CTD dimer interface is retained in the assembled CA. Furthermore, molecular docking revealed an additional, novel CTD trimer interface.

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Figures

Fig. 1
Fig. 1
Coomassie Blue-stained SDS-PAGE of puri fied HIV-1 CA and CA mutants before assembly (t) and following assembly and centrifugation (s and p). Assembled CA tubes are present in the pellet (p) after high-speed centrifugation.
Fig. 2
Fig. 2
EM of negatively stained CA wt and CA mutant assemblies. (a) CA wt assemblies show bundled tubes. (b) CA E45A assembles more efficiently, but with multiple layers. (c) CA A92E assembles single, long, and well-ordered tubes. Scale bars, 100 nm.
Fig. 3
Fig. 3
CryoEM micrographs of recombinant HIV-1 CA tubular assemblies. (a) CA wt tubes. (b) CA A92E tubes. Scale bars, 100 nm. (Reproduced from ref. with permission from Elsevier Science).
Fig. 4
Fig. 4
Indexing of HIV-1 CA helical tubes. (a) The geometry of a discontinuous helical structure. A motif (circle) is repeated with a vertical spacing p along a helix of pitch P. (b) The diffraction pattern of the helical structure shown in (a). Sets of cross-like intensities repeated with a vertical spacing of 1/p. There are two principal spacings: 1/P within any single X pattern and 1/p between the centers of separate X patterns. All layer lines are separated by 1/c. (c) An HIV-1 CA A92E tube image. Scale bars, 40nm. (d) The Fourier transform of (c) with helical indices. The arrow at the top points to the layer lane at 20 Å resolution.
Fig. 5
Fig. 5
Initial reconstruction using IHRSR. (a) Helical symmetry determination for each iterative cycle. Δφ and Δz, starting from the initial values, converge to the stable values after ten iterative re finement cycles. (b) The initial map using IHRSR.
Fig. 6
Fig. 6
CTF determination using ctfit program. The parameters of the CTF in each micrograph were determined by fitting of the intensity of CTF to the power spectrum calculated from segmented tubes. Shown are a power spectrum (thick line) and fitted CTF (thin line) of tubes recorded at a defocus value of 2.5 μm.
Fig. 7
Fig. 7
Flow chart for the refinement of the 3D map. The CTF-corrected segments are aligned against the reference projections and back-projected to generate a 3D volume. The helical symmetry was then imposed onto the 3D volume, which is then used as the new reference for the next refinement cycle. The cycle (dashed box) is iterated until a stable reconstruction is obtained.
Fig. 8
Fig. 8
The 3D density map of the A92E CA tubes from the (−13, 11) helical family. (a – c) The density map of CA tubes is displayed as three orthogonal slices: parallel to the tube axis and close to the surface (a), perpendicular to the tube axis (b), and parallel to and through the tube axis (c). Scale bars, 10 nm. (d) Surface rendering of the 3D density map contoured at 2.3σ (solid) and 1.3σ (transparent) enclosing 65% and 100% volume, respectively. Dashed lines connect hexamers in the three distinct helical arrangements denoted as n = −2, 11, and −13 helices; n is the Bessel order and the sign indicates the handedness of the helix (−sign as left hand, and +sign as right hand). (e) Fourier shell correlation and phase residual plots of the 3D density map reconstructed from 197 tubular segments. The resolution of the map is 16 Å at FSC = 0.5, or at phase residue of 65°. (Reproduced from ref. with permission from Elsevier Science).
Fig. 9
Fig. 9
Molecular Docking of NTD and CTD domain models independently into the tubular density map (contour enclosing 90% volume). (a) An overlay of docked pseudo-atomic model and 3D density map viewed from the tube surface. (b, c) Slab views to show the model fitting at NTD region (b) and the CTD region (c). Three docked CA-hexamers are displayed. The local threefold axis is marked by asterisk. (Reproduced from ref. with permission from Elsevier Science).
Fig. 10
Fig. 10
Novel CTD–CTD interactions at the local threefold axis in CA tubular assemblies. (a) A pseudo-atomic model of three CA-hexamers in the assembled tube. Boxed region encloses the new CTD–CTD interface at the local threefold axis (indicated by asterisk). (b) A detailed view of the boxed region illustrating the interactions at the interface. Side chains of K203, P207, E213, T216, and Q219 are shown in ball and stick representation. Mutations of these residues are known to affect in vitro assembly and capsid stability. A pair of spatially close residues, P207/T216, were tested by cysteine cross-linking (20). (Reproduced from ref. with permission from Elsevier Science).
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